Genome sequencing has revolutionized biology and medicine. A 5-fold decrease in sequencing cost over the past 10 years has fueled an explosive growth in the availability of genome sequence data for numerous organisms. Despite these advances, the vast majority of the value from sequence data has yet to be realized, as the cost of routine sequencing is prohibitive. Current sequencing technologies based on capillary electrophoresis will likely not allow order-of-magnitude decreases in cost. Alternative sequencing technologies are required. Here we propose to use DNA polymerase enzyme as a fast and frugal sequencing engine by monitoring DNA polymerization in real-time. Nanofluidics, Inc. was established as a spin-out from Cornell University explicitly to leverage 2 technological advances that enable real-time single-molecule sequencing system. The first is an optical confinement technology, the zero-mode waveguide (ZMW), which allows detection of single nucleotide incorporation in real-time during processive DNA polymerization. The second, terminal-phosphate fluorescent labeling, is a method of attaching fluorophores to nucleotides such that they are automatically removed from the DNA strand after incorporation. By leaving the DNA structure un-hindered with fluorophores, this method allows highly processive incorporation even using 100% replacement with labeled nucleotides. The combination of these technologies eliminates the need for slow and expensive washing of the reaction or un-blocking of the polymerase. Because the polymerase is free-running, the sequence read can proceed as long as the polymerase continues synthesizing, which can be as long as hundreds of thousands of bases. Both the ZMW and the polymerase are small, and the system has no fluidics or moving parts, making the technology amenable to high degrees of multiplexing. The goal of this program is to deploy these technologies in a 4-color, real-time, multiplex single-molecule DNA sequencing system that will enable sequencing of a mammalian genome for $50,000 by 2008, and $1000 by 2010.